Insertion and Characterization of the cry1Ia1 Gene in the Potato Cultivar Spunta for Resistance to Potato Tuber Moth

Abstract

<jats:p>Potato tuber moth (<jats:italic>Phthorimaea operculella</jats:italic>) is a serious pest of potatoes in tropical and subtropical regions of the world, including South Africa. The <jats:italic>cry1Ia1</jats:italic> gene (from <jats:italic>Bacillus thuringiensis</jats:italic>) under the control of the 35S cauliflower mosaic virus promoter was transformed into the potato (<jats:italic>Solanum tuberosum</jats:italic>) cultivar Spunta to develop a cultivar with resistance to potato tuber moth for release in South Africa. Two transformation events, ‘SpuntaG2’ and ‘SpuntaG3’, were selected and subjected to extensive molecular analyses as required by the regulatory agencies of South Africa. Southern hybridization experiments indicated that ‘SpuntaG2’ and ‘SpuntaG3’ had one and three copies of the <jats:italic>cry1Ia1</jats:italic> gene, respectively, and that the gene insertion was stable through multiple clonal generations. Furthermore, the sequence of the <jats:italic>cry1Ia1</jats:italic> gene in ‘SpuntaG2’ was compared with the known sequence of the <jats:italic>cry1Ia1</jats:italic> gene and found to be identical. Polymerase chain reaction (PCR) amplification using primers for plasmid “backbone” genes demonstrated that ‘SpuntaG2’ contained no backbone plasmid genes, whereas ‘SpuntaG3’ contained several backbone plasmid genes. Therefore, further analyses were limited to ‘SpuntaG2’, and event-specific primers were developed for this cultivar. Analysis of the left and right border regions in ‘SpuntaG2’ demonstrated that the insertion of the <jats:italic>cry1Ia1</jats:italic> gene did not disrupt any functional genes nor did it create new open reading frames that encoded proteins with a significant match to the non-redundant sequence database queried by the BLASTP program. Enzyme-linked immunoabsorbent assays (ELISA) tests indicate that the <jats:italic>cry1Ia1</jats:italic> gene was expressed at a mean concentration of 2.24 μg·g<jats:sup>−1</jats:sup> fresh weight in leaf tissue and 0.12 μg·g<jats:sup>−1</jats:sup> fresh weight in tubers. This study demonstrates the extensive molecular characterization that is necessary to apply for deregulation of a genetically modified crop and these data have been used in a regulatory package for the general release of ‘SpuntaG2’.</jats:p>

Safety Evaluations of the Cry1Ia1 Protein Found in the Transgenic Potato ‘SpuntaG2’

“…The acute toxicity test established a no adverse effect level (NOAEL) of 2000 mgÁkg -1 body weight per day, from which an ADI of 20 mgÁkg -1 body weight per day was calculated (Benford, 2000). Using the expression levels of Cry1Ia1 in 'SpuntaG2' tubers [0.12 mgÁg -1 fresh weight (Zarka et al, 2010)], the expected dose of Cry1Ia1 consumed by the South African population can be estimated. The South African Medical Research Council (2002) estimates average adult consumption of potatoes at 7.9 gÁkg -1 body weight per day.…”

“…In a previous study, 'SpuntaG2', a potato cultivar containing the cry1Ia1 gene, was extensively characterized at the molecular level (Zarka et al, 2010). The results indicated that the cry1la1 gene in 'SpuntaG2' was unchanged during the transformation process, was stably integrated into the potato 10 1 0 0 1 1 1 2 10 2 0 0 1 2 2 2 100 1 0 0 1 2 2 1 100 2 0 0 1 2 2 2 0 1 2 2 1 2 z Each well contained 1mL of diet, the designated amount of Cry1Ia1 protein, and two newly hatched M. sexta larvae.…”

“…Furthermore, sequence analysis of the protein revealed no significant match with known toxins except for other Cry insecticidal proteins. Acute toxicity tests from our study and ELISA data from Zarka et al (2010) allowed us to calculate an ADI for the Cry1Ia1 protein. Assuming a diet including only 'SpuntaG2' potatoes, the EDI for the Cry1Ia1 protein was more than 22,000 times less than the ADI.…”

“…The 29-kDa neomycin phosphotransferase enzyme inactivates aminoglycoside antibiotics by transferring the g-phosphate from ATP to the respective antibiotic. A complete characterization of the 'SpuntaG2' transgenic event has been conducted (Zarka et al, 2010) and they showed that there is a single copy of each gene with no interruption of endogenous genes during the transformation process. That article is the first one in a three-article series and contains a complete research rationale.…”

Evaluations of Transgenic Potatoes for Resistance to Potato Tuberworm in the Laboratory and Field

“…Plant Material The potato variety 'Spunta' was transformed using vector constructs designed to constitutively express the cry1Ia1 gene as described in Li et al (1999) and Zarka et al (2010). Tubers of these transformed Spunta lines ('SpuntaG2', 'SpuntaG3' and 'Spunta6a3') were produced under field conditions by the Michigan State University Potato Breeding Program and held at 3°C prior to use in bioassays.…”